Many industrially important microorganisms use glucose as their main carbon source to produce biosynthetic products. Therefore, cost-effective and efficient biosynthetic production of these products require that a carbon source, such as glucose be converted to said products at a high percentage yield. To meet this need, it would be advantageous to increase the influx of carbon sources into and through various metabolic pathways, such as the common aromatic pathway, the tricarboxylic acid (TCA) pathway, and the anaplerotic oxaloacetate synthetic pathway.
In the initial stage of host cell carbohydrate metabolism, each glucose molecule is converted to two molecules of phosphoenolpyruvate (PEP) in the cytosol. PEP is one of the major metabolic building blocks that cells use in their biosynthetic as routes. For example, PEP may be further converted to pyruvate and chemical reactions that convert glucose to pyruvate are referred to as the Embden-Meyerhoff pathway. All of the metabolic intermediates between the initial glucose carbohydrate and the final product, pyruvate, are phosphorylated compounds. Bacteria, which ferment glucose through the Embden-Meyerhof pathway, such as members of Enterobacteriacea and Vibrionaceae, are described in Bouvet et al., (1989) International Journal of Systematic Bacteriology, 39:61-67. Pyruvate may then by metabolized to yield products such as lactate, ethanol, formate, acetate and acetyl CoA. (See FIGS. 1A and 1B).
In addition to the Embden-Meyerhof pathway, many bacteria posses an active transport system known as the phosphoenolpyruvate (PEP)—dependent phosphotransferase transport system (PTS). This system couples the transport of a carbon source, such as glucose to its phosphorylation. The phosphoryl group is transferred sequentially from PEP to enzyme I and from enzyme I to protein HPr. The actual translocation step is catalyzed by a family of membrane bound enzymes (called enzyme II), each of which is specific for one or a few carbon sources. Reference is made to Postma et al., (1993) Phosphoenolpyruvate: Carbohydrate Phosphotransferase Systems in Bacteria, Microbiol. Reviews. 57:543-594 and Postma P. W. (1996) Phosphotransferase System for Glucose and Other Sugars. In: Neidhardt et al., Eds. Escherichia coli AND Salmonella typhimurium: CELLULAR AND MOLECULAR BIOLOGY. Vol. 1. Washington, D.C. ASM Press pp 127-141. However, due to the fact that PTS metabolizes PEP to phosphorylate the carbon source, the PTS system decreases the efficiency of carbon substrate conversion to a desired product. In glycolysis, two molecules of PEP are formed for every molecule of glucose catabolized. However, one molecule of PEP is required for PTS to function, leaving only one molecule of PEP available for other biosynthetic reactions.
Due to the role of PEP as a central metabolite, numerous approaches have been utilized to increase PEP supply in the cell and some of these are listed below:
a) eliminating pyruvate kinase activity by producing pyk mutants. Pyruvate kinase catalyzes the conversion of PEP to pyruvate. (Mori et al., (1987) Agric. Biol. Chem. 51:129-138);
b) eliminating PEP carboxylase activity by producing ppc mutants. PEP carboxylase catalyzes the conversion of PEP to oxaloacetate. (Miller et al., (1987) J. Ind. Microbiol. 2:143-149);
c) amplifying the expression of pps which encodes PEP synthase. PEP synthase catalyzes the conversion of pyruvate to PEP (U.S. Ser. No. 08/307,371); and
d) increasing the supply of D-erythrose-4-phosphate (E4P) by for example overexpression of a transketolase gene (tktA or tktB) (U.S. Pat. No. 5,168,056) or overexpression of the transaldolase gene (talA) (lida et al., (1983) J. Bacteriol. 175:5375-5383). Transketolase catalyzes the conversion of D-fructose-6-phosphate to E4P and transaldolase catalyzes the conversion of D-sedoheptulose-7-phosphate plus glyceraldehyde-3-phosphate to E4P plus fructose-6-phophate.
In addition to the above listed approaches, researchers have looked at methods of decreasing PTS:PEP dependent consumption by eliminating or modifying the function of the PTS. This approach is also attractive because PEP is twice as energetic as ATP. Many of these efforts focus on using an inactive PTS system. Examples of studies manipulating the PTS system include                a) restoring a glucose phenotype (Glu+) in PTS inactivated E. coli cells by introducing the genes glf and glk which encode a glucose-facilitated diffusion protein and glucokinase, respectively, from Zymomonas mobilis, wherein the E. coli cells have an inactivated PTS due to a deletion of the pstHlcrr operon (U.S. Pat. No. 5,602,030 and Snoep et al., (1994) J. Bact. 176:2133-2135) and        b) subjecting PTS−/Glu− E. coli strains to continuous culture selection on glucose and obtaining Glu+ revertants (PTS−/Glu+) with the capacity to obtain growth rates similar or higher than that of wild-type PTS−/Glu− strains. (Flores et al., (2002) Metab. Eng 4:124-137; Flores et al., (1996) Nature Biotechnol. 14:620-623 and WO96/34961).        
However, these approaches have various limitations. In general, the use of heterologous genes does not always work efficiently in new hosts. Additionally, membrane proteins, such as a glucose-facilitated diffusion protein, are usually intimately associated with lipids in the cell membrane and these can vary from species to species. Introduced soluble proteins such as glucokinase, may be subject to protease degradation. Further the use of spontaneous mutations in a cell to regain a phenotype can have unpredictable outcomes, and for industrial processes it is desirable to use completely characterized strains.
Contrary to the methods previously described, the present invention increases carbon flow to metabolic pathways in bacterial strains capable of transporting glucose without consuming PEP during the process. The conserved PEP or PEP precursors can then be redirected into a given metabolic pathway for enhanced production of a desired product. These strains are generated in cells having an inactivated PEP-dependent PTS by modifying an endogenous chromosomal regulatory region that is operably linked to a glucose assimilation protein and more specifically to a glucose transporter and/or a glucose phosphorylating protein, to restore or re-attain the ability of the cell to use glucose as a carbon source while maintaining an inactivated PTS. These cells are designated PTS−/Glu+.